Phosphoprotein Array Research Articles

Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling

Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain ‘mini-myoferlin’. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate ‘mini-myoferlin’ can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.

CCDC88A, a prognostic factor for human pancreatic cancers, promotes the motility and invasiveness of pancreatic cancer cells.

BackgroundCoiled-Coil Domain Containing 88A (CCDC88A) was identified as a substrate of the serine/threonine kinase Akt that is capable of binding to the actin cytoskeleton. The aim of this study was to investigate the potential role of CCDC88A in the migration and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells.MethodsImmunohistochemistry was performed to determine whether high CCDC88A expression in human PDAC tissues is correlated with poor prognosis. Immunoprecipitation, immunoblotting and immunocytochemistry were performed to determine the intracellular distribution of CCDC88A, and its association with the serine/threonine kinase Akt and actin-filaments in PDAC cells. Phosphoprotein array analysis was performed to determine CCDC88A-associated intracellular signaling pathways. Finally, immunofluorescence analyses and Matrigel invasion assays were performed to examine the effects of CCDC88A on the formation of cell protrusions and PDAC cell invasion.ResultsExpression of CCDC88A in PDAC tissue was significantly correlated with overall survival. CCDC88A was co-localized with peripheral actin structures in cell protrusions of migrating PDAC cells. Knockdown of CCDC88A inhibited the migration and invasiveness of PDAC cells through a decrease in cell protrusions. Although CCDC88A has been previously reported to be a binding partner and substrate of Akt, the level of active Akt was not associated with the translocation of CCDC88A towards cell protrusions. CCDC88A-dependent promotion of cell migration and invasiveness was not modulated by Akt signaling. Knockdown of CCDC88A decreased phosphorylated Src and ERK1/2 and increased phosphorylated AMPK1 in PDAC cells. Knockdown of AMPK1 inhibited the migration and invasiveness of PDAC cells. The combined data suggest that CCDC88A may be a useful marker for predicting the outcome of patients with PDAC and that CCDC88A can promote PDAC cell migration and invasion through a signaling pathway that involves phosphorylation of Src and ERK1/2 and/or dephosphorylation of AMPK1.ConclusionsCCDC88A was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and increased the migration and invasiveness of PDAC cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0466-0) contains supplementary material, which is available to authorized users.

Vav3 is linked to poor prognosis of pancreatic cancers and promotes the motility and invasiveness of pancreatic cancer cells

Background/objectivesThe aim of this study was to investigate the role of the guanine nucleotide exchange factor Vav3 in the motility and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells. MethodsImmunohistochemistry was used to determine whether high Vav3 expression in human PDAC tissues is correlated with poor prognosis. Immunocytochemistry was used to determine the association and intracellular distribution of Vav3, Rac1 and Akt in PDAC cells. Phosphoprotein array analysis was performed to determine the Vav3-associated intracellular signaling pathways. Immunocytochemistry and Matrigel invasion assays were used to examine the effects of Vav3 on the formation of cell protrusions and PDAC cell invasion. ResultsExpression of Vav3 in PDAC tissue was significantly correlated with overall survival. Vav3 was localized in cell protrusions of migrating PDAC cells. Knockdown of Vav3 inhibited the motility and invasiveness of PDAC cells through a decrease in cell protrusions. The levels of active Rac1 or active Akt were not associated with the concentration of Vav3 in cell protrusions. The Vav3-dependent promotion of motility and invasiveness was not modulated by Rac1 or Akt. Additionally, knockdown of Vav3 increased phosphorylated WNK1 in PDAC cells, and knockdown of WNK1 inhibited the motility and invasiveness. This study suggests that Vav3 can be a useful marker for predicting the outcome of patients with PDAC and that Vav3 can promote PDAC cell motility and invasion through association with dephosphorylation of WNK1. ConclusionsVav3 was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and thereby increased the motility and invasiveness of PDAC cells.

Chromatin remodeling gene AT-rich interactive domain-containing protein 1A suppresses gastric cancer cell proliferation by targeting PIK3CA and PDK1

The tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) was frequently mutated in cancers. The modulation mechanism of ARID1A for PI3K/AKT signaling in gastric cancer (GC) remains elusive. Here, we found that depletion of endogenous ARID1A enhanced the in vitro proliferation, colony formation, cellular growth, nutrient uptake and in vivo xenograft tumor growth of GC cells. PI3K/AKT activation by ARID1A-silencing was profiled using a phospho-protein antibody array. The phosphorylation of PDK1, AKT, GSK3β and 70S6K, and the protein and mRNA expressions of PI3K and PDK1, were upregulated by ARID1A-silencing. Chromatin immunoprecipitation and luciferase reporter assay revealed that ARID1A-involved SWI/SNF complex inhibited PIK3CA and PDK1 transcription by direct binding to their promoters. Serial deletion mutation analyses revealed that the ARID1A central region containing the HIC1-binding domain, but not the ARID DNA-binding domain and the C-terminal domain, was essential for the inhibition of GC cell growth, PI3K/AKT pathway phosphorylation and its transcriptional modulation activity of PIK3CA and PDK1. The proliferation, cellular growth and glucose consumption of ARID1A-deficient GC cells were efficiently prohibited by allosteric inhibitors mk2206 and LY294002, which targeting AKT and PI3K, respectively. Both inhibitors also downregulated the phosphorylation of PI3K/AKT pathway in ARID1A-deficient GC cells. Such cells were sensitized to the treatment of LY294002, and AT7867, another inhibitor of AKT and p70S6K. The administration of LY294002 alone inhibited the in vivo growth of ARID1A- deficient GC cells in mouse xenograft model. Our study provides a novel insight into the modulatory function and mechanism of ARID1A in PI3K/AKT signaling in GC.

Fibroblast Growth Factor 23 drives MMP13 expression in human osteoarthritic chondrocytes in a Klotho-independent manner

Fibroblast Growth Factor 23 (FGF23) may represent an attractive candidate that could participate to the osteoarthritic (OA)-induced phenotype switch of chondrocytes. To address this hypothesis, we investigated the expression of FGF23, its receptors (FGFRs) and co-receptor (Klotho) in human cartilage and studied the effects of rhFGF23 on OA chondrocytes. Gene expression or protein levels were analysed by RT-PCR and immunohistochemistry. Collagenase 3 (MMP13) activity was measured by a fluorescent assay. MAPK signalling pathways were investigated by phosphoprotein array, immunoblotting and the use of selective inhibitors. RNA silencing was performed to confirm the respective contribution of FGFR1 and Klotho. We showed that the expression of FGF23, FGFR1 and Klotho was up-regulated at both mRNA and protein levels in OA chondrocytes when compared to healthy ones. These overexpressions were markedly elevated in the damaged regions of OA cartilage. When stimulated with rhFGF23, OA chondrocytes displayed an extended expression of FGF23 and of markers of hypertrophy such as MMP13, COL10A1, and VEGF. We demonstrated that FGF23 auto-stimulation was both FGFR1-and Klotho-dependent, whereas the expression of markers of hypertrophy was mainly dependent on FGFR1 alone. Finally, we showed that FGF23-induced MMP13 expression was strongly regulated by the MEK/ERK cascade and to a lesser extent, by the PI-3K/AKT pathway. These results demonstrate that FGF23 sustains differentiation of OA chondrocytes in a Klotho-independent manner.

BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma.

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.

Aberrant Activation of the PI3K/mTOR Pathway Promotes Resistance to Sorafenib in AML

Therapy directed against oncogenic FLT3 has been shown to induce response in patients with AML, but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant MV4-11 and MOLM-13 cell lines by sustained treatment with the FLT3 inhibitor sorafenib. MV4-11 cells express only FLT3-ITD, while MOLM-13 cells express wild-type FLT3 and FLT3-ITD. Both cell lines are dependent on FLT3 activation as sorafenib, PKC-412, and AC220, but not imatinib, dasatinib, nilotinib or bosutinib, inhibit cell survival in both cell lines. After treatment with sorafenib for 90 days, we observed that both cell lines displayed resistance to sorafenib as well as to AC220 suggesting that sustained treatment with an FLT3 inhibitor results in acquired resistance to multiple FLT3 inhibitors.To test whether sorafenib was still effective in FLT3 inhibition, we treated sensitive and resistant cells with sorafenib or DMSO and then stimulated with FLT3 ligand (FL). While DMSO-treated resistant cells responded to FL as expected, sorafenib-treated cells displayed poor FLT3 activation, suggesting that sorafenib was still capable of inhibiting FLT3 activation in the resistant cells. Surprisingly we observed that the resistant cells treated with DMSO had a much more robust response to ligand in terms of FLT3 activation. Similar results were observed with AKT and ERK activation, as sorafenib-treated cells poorly responded to FL-stimulation. Furthermore, resistant cells treated with sorafenib could still form colonies similar to DMSO-treated cells indicating that these cells were no longer dependent on FLT3 activation, although sorafenib could partially block FLT3 activation.To determine whether secondary mutations occurred in FLT3, we sequenced the whole coding region using Sanger sequencing. Except for a mutation in the extracellular domain in all four cell lines no mutations were detected in the inhibitor binding site. Similar to Sanger sequencing, mass spectroscopic analysis of affinity-enriched FLT3 indicated no differences in the intracellular part of FLT3 between sensitive and resistant cells. Because we observed an unexpected activation of FLT3 in resistant cells stimulated with FL, we hypothesized that certain FLT3 residues remain hyper-tyrosine phosphorylated. To test that we used phospho-specific antibodies against known FLT3 tyrosine phosphorylation sites. Although we observed an increase in total FLT3 tyrosine phosphorylation, we were unable to identify a single site that was selectively hyper-phosphorylated. Instead, all sites remained slightly more phosphorylated compared to control cells.Gene expression analysis of mRNA from sensitive and resistant cell lines using ANOVA and significance analysis of microarrays (SAM) suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype. In addition to pathway enrichment, using a phospho-protein antibody array, we found that phosphorylation of the mTOR substrates S6K and AKT were selectively increased in resistant cells. We also observed an increase in STAT3 phosphorylation. Elevated STAT3 phosphorylation was probably due to the previously described upregulation of JAK3 expression in sorafenib-resistant AML. We also observed enrichment of an mTOR signature in AML blasts from eight patients with sorafenib-resistant AML (GSE35907) and in AML blasts from patients expressing FLT3-ITD compared to those lacking FLT3-ITD (525 samples, GSE14468).Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in the resistant cell lines. Treatment of cells with a higher concentration of gedatolisib did not alter the phosphorylation of other signaling proteins except for AKT and S6K which was detected by a phospho-specific antibody array. The array data was further verified with western blotting using phospho-specific antibodies against AKT, ERK1/2, p38 and S6K. These results suggest that gedatolisib efficiently blocks the downstream effectors PI3K/mTOR without affecting other signaling pathways.Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD dependent AML results in resistance to drugs targeting FLT3 and treatment with selective PI3K/mTOR inhibitors are likely to be effective in overcoming acquired resistance in AML patients. DisclosuresStegmaier:Novartis Pharmaceuticals: Consultancy.

Abstract LB-C22: Acquired resistance to the cMET inhibitor savolitinib in lung cancer models through EGFR/mTOR/MYC deregulation and adoption of PIM signaling

Abstract Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. Aberrant receptor tyrosine kinase (RTK) signaling is a well-documented driver of disease onset and progression in multiple cancer types, including non-small cell lung cancer (NSCLC), where the cMET RTK contributes to tumor progression, maintenance and resistance to targeted therapies. Here, we explore the therapeutic potential of the potent and selective cMET inhibitor savolitinib (volitinib, AZD6094, HMPL-504) in NSCLC and begin to elucidate mechanisms of acquired savolitinib resistance in preclinical models. Using in vitro proliferation assays and immunoblot analysis, we determine that savolitinib rapidly inhibits cMET auto-phosphorylation/activation and reduces the viability of NSCLC cell lines NCI-H1993 and EBC-1 with a GI50 of 4.20 nM and 2.14 nM, respectively. In vivo, once daily treatment of NCI-H1993 xenografts with 3.0 mg/kg savolitinib significantly slows tumor growth, whereas treatment of EBC-1 xenografts with 30.0 mg/kg results in tumor stasis. Importantly, we observe tumor regressions in a patient-derived xenograft model of a NSCLC lymph node metastasis, HLXF-036LN, dosed with savolitinib 50.0 mg/kg once daily. Pharmacodynamic analysis of in vitro and in vivo models shows that savolitinib sensitivity correlates with blockade of PI3K/AKT and MAPK signaling, and interestingly, with cMYC (MYC) protein down-regulation. To elucidate mechanisms of acquired resistance in NSCLC, we generated savolitinib resistance in vitro using the NCI-H1993 and EBC-1 cell lines and further sub-cloned resistant NCI-H1993 cells to study the heterogeneity of resistance mechanisms. Using small-molecule screening, phospho-protein arrays and interrogation of signaling pathway activity by immunoblot, we identify 1) deregulated mTORC1/2 signaling and 2) the uncoupling of MYC expression from cMET activation as commonly contributing to resistance in all clones tested. RNA interference (siRNA) and MYC over-expression experiments confirm the novel finding that sustained MYC expression can partially drive resistance to a tyrosine kinase inhibitor such as savolitinib. Additionally, we identify clone-specific resistance mechanisms arising via a previously-described switch to EGFR dependence or by our novel finding of a de novo requirement for PIM signaling. Taken together, this work demonstrates the preclinical efficacy of savolitinib in NSCLC and provides an initial characterization of potential resistance mechanisms, identifying core resistance targets and clone-specific vulnerabilities that could be exploited to counter acquired savolitinib resistance that may emerge in the clinic. Citation Format: Ryan E. Henry, Evan R. Barry, Brendon Ladd, Aleksandra Markovets, Garry J. Beran, Yongxin Ren, Feng Zhou, Lillian Castriotta, Ammar Adam, Weiguo Qing, Weiguo Su, Edwin Clark, Celina M. D'Cruz, Alwin Schuller. Acquired resistance to the cMET inhibitor savolitinib in lung cancer models through EGFR/mTOR/MYC deregulation and adoption of PIM signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C22.

Abstract LB-B19: BKM-120: a phosphatidyl-inositol-3 kinase inhibitor with anti-migratory properties in glioblastoma

Abstract Glioblastoma (GBM) is the most common glial brain tumor and is also one of the most lethal human cancers: patients have a median survival of 15 months with a five-year survival rate of only 3%. The current standard-of-care has remained the same for the last decade and consists of maximal safe surgical resection followed by radio- and chemotherapy. GBM cells are highly infiltrative, leading to invasion of normal brain tissue by tumor cells. These invasive tumor cells render GBM a surgically incurable disease and tumor recurrence is almost inevitable, with 90% of patients developing new lesions within 2-3 cm of the original site or at distant sites in the brain. Moreover, invasion may increase further during anti-angiogenic therapy with bevacizumab and no anti-invasive approaches are yet available clinically. The phosphatidyl-inositol-3 kinase (PI3K) pathway is frequently deregulated in cancer and is activated in the majority of GBM cases due to constitutive receptor tyrosine kinase activation as well as inactivating mutations/deletions of PTEN (33%) or activating PI3K mutations (17%). In addition PI3K plays a role in cell migration in some cell types and a number of small molecule PI3K inhibitors are under investigation in oncology clinical trials. BKM-120 (Buparlisib), is a CNS-penetrant selective pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for several types of solid tumor, including GBM. We initially observed that BKM-120 is a potent anti-invasive molecule in GBM cell lines and patient-derived glioma stem-like cells in vitro. The anti-migratory effects of BKM-120 were clearly distinguishable from cytostatic and cytotoxic effects that occurred at higher drug concentrations and after a longer duration of drug exposure. The blockade of migration was reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenograft model. Mechanistically we found that GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. However, BKM-120 substantially reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition may contribute to the anti-invasive effects of BKM-120. Overall, our data suggest that the CNS-penetrant PI3K inhibitor BKM-120 may be a useful anti-migratory drug for the treatment of highly invasive tumors such as glioblastoma. Citation Format: Maria Carmela Speranza, Michal O. Nowicki, Choi-Fong Cho, E. Antonio Chiocca, Sean E. Lawler. BKM-120: a phosphatidyl-inositol-3 kinase inhibitor with anti-migratory properties in glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B19.

CBIO-44RESTING MEMBRANE POTENTIAL CONTROLS STEM CELL STATE AND DIFFERENTIATION OF GLIOBLASTOMA CANCER STEM CELLS

Glioblastoma (GBM) is the most common malignant brain tumor in adults with a poor prognosis despite aggressive treatment. Resting membrane potential (RMP) has been shown to be an important signaling mechanism in physiological stem cells. Further the importance of ion channels and RMP have been studied in glioma. We evaluated the relevance of RMP in GBM in the context of the cancer stem cell(CSC) hypothesis. Undifferentiated GBM CSCs were depolarized compared to differentiated counterparts [RMP of -5 +/- 4 (n = 33) -24 + /- 20 (n = 31, P < 0.001)]. Depolarization using exogenous potassium (KCl) of GBM CSCs grown under differentiating conditions was sufficient to prevent differentiation and enhance self renewal when compared to osmotic controls (NaCl). Hyperpolarization using genetic (mutant TREK transduction) and pharmacological (omeprazole and salinomycin) was sufficient to induce differentiation and limit self renewal in GBM CSCs grown under undifferentiated conditions. To evaluate for a mechanism mediating this phenomenon we performed a phospho-protein array comparing the undifferentiated GBM CSCs and those treated with omeprazole or salinomycin. We noted upregulation of phospho-proteins related to senescence pathways in treated cells but no specific key pathway. Finally, this evidence suggests that RMP may be an important regulator of stem cell state of GBM CSCs. Further it may be a target of novel treatment strategies.

STEM-14CELLULAR RESTING TRANSMEMBRANE POTENTIAL INSTRUCTS DIFFERENTIATION STATE OF GBM STEM-LIKE CELLS

INTRODUCTION: Glioblastoma (GBM) is the most common malignant brain tumor in adults with a poor prognosis despite maximal treatment. Resting membrane potential (RMP) has been shown to be an important regulator of the differentiation state of embryonic and somatic stem cells. We evaluated the relevance of RMP in GBM stem-like cells (GSC). METHODS: Whole cell patch clamp technique was used to determine the RMP. RESULTS: Undifferentiated GSC had a RMP of -5mV +/-4 (n = 33) compared to their differentiated counterparts, which had a mean RMP of -24mV +/-20 (n = 31, P < 0.001). To determine whether the cellular RMP played an instructive role in the differentiation state of GSC we used pharmacologic and genetic approaches to alter the potential. We added additional potassium chloride (KCl) to differentiated GCS in order to induce depolarization, and compared to osmotic controls using sodium chloride (NaCl). We found that GSC exposed to high KCl retained stem markers and sphere forming capacity compared to controls. We then examined whether hyperpolarization was sufficient to induce differentiation. We treated GSC with vacuolar ATPase inhibitors and salinomycin (potassium ionophore), which induced relative hyperpolarization of GSC and induction of differentiation. Transduction of GSC with inducible potassium ion channel led to hyperpolarization and differentiation. To identify potential mechanisms coupling electrical alterations to biochemical signal transduction pathways we performed extensive phospho-protein array studies comparing the undifferentiated GSC and cells hyperpolarized with omeprazole or salinomycin. Attenuation of key developmental and pro-growth signaling pathways was observed. CONCLUSIONS: Alteration of RMP may represent a new therapeutic strategy.

Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis.

BackgroundVarious signals are known to participate in the pathogenesis of lung fibrosis. Our aim was to determine which signal is predominantly mobilized in the early inflammatory phase and thereafter modulates the development of lung fibrosis.MethodsMice received a single dose of 3 mg/kg body weight of bleomycin (BLM) and were sacrificed at designated days post-instillation (dpi). Lung homogenates and sections from mice in the early inflammatory phase were subjected to phospho-protein array analysis and immunofluorescence studies, respectively. Bronchoalveolar lavage fluid (BALF) from mice was subjected to an enzyme-linked immunosorbent assay (EIA) for interleukin (IL)-6 and evaluation of infiltrated cell populations. The effects of endogenous and exogenous IL-6 on the BLM-induced apoptotic signal in A549 cells and type 2 pneumocytes were elucidated. In addition, the effect of IL-6-neutralizing antibody on BLM-induced lung injury was evaluated.ResultsPhospho-protein array revealed that BLM induced phosphorylation of molecules downstream of the IL-6 receptor such as Stat3 and Akt in the lung at 3 dpi. At 3 dpi, immunofluorescence studies showed that signals of phospho-Stat3 and -Akt were localized in type 2 pneumocytes, and that BLM-induced IL-6-like immunoreactivity was predominantly observed in type 2 pneumocytes. Activation of caspases in BLM-treated A549 cells and type 2 pneumocytes was augmented by application of IL-6-neutralizing antibody, a PI3K inhibitor or a Stat3 inhibitor. EIA revealed that BLM-induced IL-6 in BALF was biphasic, with the first increase from 0.5 to 3 dpi followed by the second increase from 8 to 10 dpi. Blockade of the first increase of IL-6 by IL-6-neutralizing antibody enhanced apoptosis of type 2 pneumocytes and neutrophilic infiltration and markedly accelerated fibrosis in the lung. In contrast, blockade of the second increase of IL-6 by IL-6-neutralizing antibody ameliorated lung fibrosis.ConclusionsThe present study demonstrated that IL-6 could play a bidirectional role in the pathogenesis of lung fibrosis. In particular, upregulation of IL-6 at the early inflammatory stage of BLM-injured lung has antifibrotic activity through regulating the cell fate of type 2 pneumocytes in an autocrine/paracrine manner.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0261-z) contains supplementary material, which is available to authorized users.

MAP3K3 expression in tumor cells and tumor-infiltrating lymphocytes is correlated with favorable patient survival in lung cancer.

MAP3K3 is involved in both the immune response and in tumor progression. Its potential biological role in vitro in lung cancer cell lines and the association of mRNA/protein expression patterns with clinical outcome of primary lung tumors were investigated in this study. Silencing MAP3K3 using siRNA in lung cancer cell lines resulted in decreased cell proliferation, migration and invasion. These effects were associated with down-regulation of the JNK, p38, AKT, and GSK3β pathways as determined using phospho-protein and gene expression array analyses. However, MAP3K3 mRNA and protein overexpression in primary lung tumors correlated significantly with favorable patient survival. Gene cluster and pathway analyses of primary tumor datasets indicated that genes positively-correlated with MAP3K3 are significantly involved in immune response rather than the cell cycle regulators observed using in vitro analyses. These results indicate that although MAP3K3 overexpression has an oncogenic role in vitro, in primary lung adenocarcinomas it correlates with an active immune response in the tumor environment that correlates with improved patient survival. MAP3K3 may potentially not only serve as diagnostic/prognostic markers for patients with lung cancer but also provide an indicator for future investigations into immunomodulatory therapies for lung cancer.

DNA microarray and signal transduction analysis in pulmonary artery smooth muscle cells from heritable and idiopathic pulmonary arterial hypertension subjects.

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular smooth muscle contraction and proliferation. Here, we analyze genome-wide mRNA expression in human pulmonary arterial smooth muscle cells (HPASMC) isolated from three control, three hereditary (HPAH), and three idiopathic PAH (IPAH) subjects using the Affymetrix Human Gene ST 1.0 chip. The microarray analysis reveals the expression of 537 genes in HPAH and 1024 genes in IPAH changed compared with control HPASMC. Among those genes, 227 genes show similar directionality of expression in both HPAH and IPAH HPASMC. Ingenuity™ Pathway Analysis (IPA) suggests that many of those genes are involved in cellular growth/proliferation and cell cycle regulation and that signaling pathways such as the mitotic activators, polo-like kinases, ATM signaling are activated under PAH conditions. Furthermore, the analysis demonstrates downregulated mRNA expression of certain vasoactive receptors such as bradykinin receptor B2 (BKB2R). Using real time PCR, we verified the downregulated BKB2R expression in the PAH cells. Bradykinin-stimulated calcium influx is also decreased in PAH PASMC. IPA also identified transcriptional factors such p53 and Rb as downregulated, and FoxM1 and Myc as upregulated in both HPAH and IPAH HPASMC. The decreased level of phospho-p53 in PAH cells was confirmed with a phospho-protein array; and we experimentally show a dysregulated proliferation of both HPAH and IPAH PASMC. Together, the microarray experiments and bioinformatics analysis highlight an aberrant proliferation and cell cycle regulation in HPASMC from PAH subjects. These newly identified pathways may provide new targets for the treatment of both hereditary and idiopathic PAH.

Abstract B04: Hras and Kras have different transformational capacity in murine models of thyroid carcinoma

Abstract Thyroid cancer is the most common endocrine malignancy, and incidences are rising. MAP kinase (MAPK) signaling has been implicated in playing a critical role in the initiation and maintenance of thyroid cancer, as evidenced by the high incidence of non-overlapping mutations of the genes encoding RET and TRK, as well as of NRAS, HRAS, KRAS and BRAF. RAS mutations are found in 40-50% of follicular carcinomas (FTC) and 20-40% of follicular adenomas (FA), as well as follicular variant of papillary cancer (FVPTC). The most common mutations associate with thyroid tumors are in HRAS and NRAS, most often in codon 61. This is in contrast to most other epithelial tumors where KRAS codon 12/13 mutations are most common. We hypothesized that Ras isoforms have different transformational potential in thyroid epithelium. To test this hypothesis, we developed mice with a thyroid-specific knock-in of oncogenic Hras combined homozygous loss of Pten (HrasG12V/PtenHom). We report that a thyroid specific knock-in of HrasG12V coupled with Pten inactivation leads to the development of FTCs and poorly differentiated thyroid cancer (PDTCs) by 47-52 weeks of age in HrasG12V/PtenHom/TPO-cre mice. In stark contrast, mice with a thyroid specific knock-in of KrasG12D and loss of Pten (KrasG12D/PtenHom/TPO-cre) develop FTCs which rapidly progress to PDTCs and anaplastic thyroid cancer with 50% lethality at 7 weeks and complete lethality by 20 weeks. These data suggest that Kras and Hras, in cooperation with PI3K signaling, likely play distinct roles in the development and progression of disease. We sought to dissect the role of Hras versus Kras activation in FTC development. We derived stable cell lines from HrasG12V/PtenHom/TPO-cre and obtained a similar line from Kras tumors to identify cellular and molecular responses to Hras versus Kras activation and their impact on tumor phenotype. Utilizing a phospho-protein expression array, we determined that Kras activation leads to increased phosphorylation of members of the p38 MAPK pathway, whereas Hras activation increases mTOR effectors. Pathway profiling indicated differences in transcript levels of MAPK effects in response to Kras versus Hras activation. KrasG12D/Ptenhom cells had increased expression of cell cycle regulators including cyclinD1, whereas extracellular matrix protein Col1a1 was unregulated in Hras-driven cells. Further, Kras-driven cells have an increased ability to form thyrospheres on low-adherence tissue culture plate. These results suggest that Kras induced tumors have a larger population of self-renewing cells and therefore may account for the increase virulence of this oncogene in thyrocytes. These studies demonstrate a clear phenotypic difference between mutant Hras and Kras in thyroid epithelium, and suggest that the increase transformative property of mutant Kras might be mediated by over-expression of proteins that increase cell proliferation and mediate transition through the cell cycle. Citation Format: Lee Ann King, Nolan West, Aime Franco. Hras and Kras have different transformational capacity in murine models of thyroid carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B04. doi: 10.1158/1557-3125.RASONC14-B04

Abstract 13433: Angiotensin II Type 2 Receptor Regulates Skeletal Myoblast Differentiation: Implications for treatment of Cachexia and Skeletal Muscle Wasting

Patients with advanced congestive heart failure (CHF) or chronic kidney disease (CKD) often have increased angiotensin II (Ang II) levels and cachexia. We previously demonstrated that Ang II infusion in rodents causes skeletal muscle wasting and decreases muscle regenerative potential via Ang II type 1 receptor (AT1R) signaling, likely contributing to cachexia in CHF and CKD. However, the potential role of Ang II type 2 receptor (AT2R) signaling in skeletal muscle physiology remains unknown. We found that AT2R expression was robustly increased in mouse skeletal myoblasts during differentiation, suggesting that the AT2R plays an important role in skeletal muscle regeneration. To test this hypothesis, we infused mice with AT2R antagonist PD123319 (PD, 30 mg/kg/d) or agonist CGP123319 (CGP, 1 μg/kg/min) during cardiotoxin (CTX)-induced muscle injury and regeneration. PD reduced the size of regenerating myofibers (727.5±54.6 and 516.0±37.0 μm2 in sham and PD, respectively, p&lt;0.05) and expression of the myoblast differentiation markers myogenin and eMyHC (56.9% and 40.2% decrease in PD, respectively. p&lt;0.01), whereas CGP had the opposite effects. siRNA mediated AT2R knockdown in mouse primary myoblasts suppressed the increase of myogenin and desmin, resulting in lowered differentiation. We analyzed changes in phosphoprotein levels in myoblasts after AT2R knockdown by phosphoprotein array and identified multiple changes, including increased phospho-ERK1/2 levels. Importantly, inhibition of ERK1/2 restored normal myoblast differentiation in the setting of AT2R knockdown, suggesting the AT2R positively regulates myoblast differentiation by reducing ERK1/2 activity. Furthermore, we found that skeletal muscle regeneration was reduced (decreased regenerating myofiber size and myogenin/desmin expression) in a mouse myocardial infarction model of CHF, concomitantly with markedly blunted increase of AT2R expression, strongly suggesting that the AT2R plays an important role in the reduction of skeletal muscle function in CHF. These data indicate that AT2R signaling positively regulates myoblast differentiation and potentiates skeletal muscle regeneration, providing a new therapeutic target in wasting disorders such as CHF and CKD.

Abstract 3525: SOX2 promotes tumor growth via activation of the PI3K/Akt/mTORC1 signaling pathway in esophageal squamous cell carcinoma

Abstract Our previous study revealed that SOX2, a master regulator during embryogenesis, is an amplification target of 3q26.3 in esophageal squamous cell carcinoma (ESCC) and that SOX2 promotes ESCC cell proliferation. To identify the mechanisms by which SOX2 promotes proliferation of ESCC cells, we assayed multiple signaling pathways activated by SOX2 using a phosphoprotein array. We determined that SOX2 activated the AKT/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Immunoblotting confirmed that SOX2 elevated levels of p-AKT and levels of p-p70S6K and p-4E-BP1 which are direct targets of mTORC1. Effects of SOX2 knockdown, including reduced levels of p-AKT and decreased ESCC cell viability, were reversed with constitutive activation of AKT with PTEN knockdown. SOX2 also promoted in vivo tumor growth of ESCC with AKT/mTORC1 activation in mouse xenografts. LY294002, a PI3K inhibitor, suppressed the ability of SOX2 to enhance tumor growth of ESCC by reducing cell proliferation. Furthermore, tissue microarray analysis showed a positive correlation between expression levels of SOX2 and p-AKT in 61 primary ESCC tumors. Our results suggest that SOX2 promotes ESCC tumor growth via activation of the PI3K/AKT / mTORC1 signaling pathway, which enhances cell proliferation. Citation Format: Yasuyuki Gen, Kohichiroh Yasui, Tomoko Kitaichi, Akira Tomie, Yoshito Ito. SOX2 promotes tumor growth via activation of the PI3K/Akt/mTORC1 signaling pathway in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3525. doi:10.1158/1538-7445.AM2014-3525

Abstract 4501: Sensitivities of kinase inhibitors of BCR signaling are correlated with the BCR signaling pathway activities in DLBCL and CLL

Abstract B-cell malignancies are hematologic disorders for which new therapeutic agents and novel treatment strategies are evolving. B cell receptor (BCR) signaling plays an important pathogenic role in diffuse large b-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). There is rapid clinical development of inhibitors targeting BCR-associated kinases such as Spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), and phosphoinositide 3-kinase (PI3K)δ. Recent clinical studies using these agents have demonstrated effectiveness and low toxicity in CLL and DLBCL patients with relapsed/refractory disease. With a great response rate, there are still a group of patients that do not respond to these therapeutic agents. The goal of this study is to investigate potential resistance mechanism(s) of BCR pathway inhibitors based on signature-signaling profiles. 21 CLL and DLBCL cell lines with different molecular features were assessed in this study to establish relevant signaling features and their association with sensitivities to BCR signaling inhibitors. First, we evaluated response of cell lines to small molecule inhibitors targeting the BCR signaling pathways (PCI-32765, R406, CAL 101, and Dasatinib). 28% (6/21) of the cell lines were resistant to PCI-32765; we found that these cells lines were also non responsive to CAL 101 and Dasatinib. Then we used phosphoflow assays and phosphoprotein arrays to profile BCR signaling in both responsive and non-responsive cell lines. The BCR signaling activities were assessed at baseline, after stimulation with IgG-IgM, and following pretreatment with phosphatase inhibitors. Interrogating pathway activity in cell lines with pervanadate, a phosphatase inhibitor, was especially revealing. After treatment with pervanadate, we observed an increase in phosphorylation status of SYK, BTK, p38, and S6 in normal B cells. However, 24% of tumor cell lines do not exhibit phosphorylation of BCR related proteins, SYK and BTK, after pervanadate treatment. Moreover, 83% (5/6) of cell lines resistant to PCI 32765 (IC50 &amp;gt; 20μM) did not respond to pervanadate, whereas only 12.5% (1/8) sensitive cell lines (IC50 &amp;lt; 2.5uM) did not respond to pervanadate. The difference observed in these cell lines indicates tumor cell lines with an abnormal BCR signaling may not be dependent upon BCR signaling for survival. More studies are being conducted to confirm this observation in patient samples and to further explore the molecular causes of the abnormal BCR signaling. We also explore combination treatments of PCI-32765, CAL-101 and other target therapy agents, which may provide useful insights for the optimal therapeutic use of these BCR signaling inhibitors in CLL and DLBCL. Overall, our results suggest that profiling of signaling network activities represents a promising approach to molecularly classifying DLBCL and CLL subtypes for target therapies. Citation Format: Joyce O. Obidi, Patricia Burke, Laura Richman, Dirk Mendel, Haifeng Bao. Sensitivities of kinase inhibitors of BCR signaling are correlated with the BCR signaling pathway activities in DLBCL and CLL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4501. doi:10.1158/1538-7445.AM2014-4501

TGFBI (βIG-H3) is a diabetes-risk gene based on mouse and human genetic studies.

Transforming growth factor beta-induced (TGFBI/βIG-H3), also known as βig-H3, is a protein inducible by TGFβ1 and secreted by many cell types. It binds to collagen, forms part of the extracellular matrix and interacts with integrins on the cell surface. Recombinant TGFBI and transgenic TGFBI overexpression can promote both islet survival and function. In this study, we generated TGFBI KO mice and further assessed TGFBI function and signaling pathways in islets. Islets from KO mice were of normal size and quantity, and these animals were normoglycemic. However, KO islet survival and function was compromised in vitro. In vivo, KO donor islets became inferior to wild-type donor islets in achieving normoglycemia when transplanted into KO diabetic recipients. TGFBI KO mice were more prone to straptozotocin-induced diabetes than the wild-type counterpart. Phosphoprotein array analysis established that AKT1S1, a molecule linking the AKT and mTORC1 signaling pathways, was modulated by TGFBI in islets. Phosphorylation of four molecules in the AKT and mTORC1 signaling pathway, i.e. AKT, AKT1S1, RPS6 and EIF4EBP1, was upregulated in islets upon TGFBI stimulation. Suppression of AKT activity by a chemical inhibitor, or knockdown of AKT1S1, RPS6 and EIF4EBP1 expression by small interfering RNA, modulated islet survival, proving the relevance of these molecules in TGFBI-triggered signaling. Human genetic studies revealed that in the TGFBI gene and its vicinity, three single-nucleotide polymorphisms were significantly associated with type 1 diabetes risks, and one with type 2 diabetes risks. Our study suggests that TGFBI is a potential risk gene for human diabetes.

BAALC Acts As a Scaffold-Like Protein In The ERK Signaling Pathway and Promotes Leukemogenesis By Facilitating MEK–mediated ERK Activation

Significance of BAALC (brain and acute leukemia, cytoplasmic) as a poor prognostic factor for patients with cytogenetically normal acute myeloid leukemia (CN-AML) and acute lymphoblastic leukemia (ALL) has now been widely established, however, its molecular mechanisms are still mostly unknown. Available evidence suggests that BAALC is a cytoplasmic protein. Based on this finding, we carried out phosphoprotein array of cytoplasmic signal transduction-related proteins and sought to find out the deregulated signaling underlying BAALC-mediated leukemogenesis. Using BAALC-overexpressed HEK293T cells, we found enhanced phosphorylation of Extracellular signal-regulated kinase (ERK), and its downstream target p90RSK. Besides, Jurkat cells, a human ALL cell line, showed sustained phosphorylation of ERK when BAALC is overexpressed. These results suggest that BAALC acts as a scaffold-like molecule for the cytoplasmic mitogen-activated protein kinase kinase (MEK), upstream ERK activator and activates ERK signaling pathway. Immunoprecipitation assay revealed that BAALC binds to MEK in HEK293T cells. Co-localization of BAALC and MEK in the cytoplasm of HEK293T was also detected by immunofluorescence experiment. The proportion of the phosphorylated form in the MEK-bound ERK was increased compared to its control, indicating that BAALC binds to MEK and directly up-regulates its kinase activity to phosphorylate ERK. BAALC accelerated cell growth and increased the phosphorylation of ERK when overexpressed in Jurkat or HEL cells. Those cells showed accelerated cell cycling with no change of apoptotic status. On the other hand, BAALC knockdown decreased cellular proliferation as well as phosphorylation of ERK in the human leukemia cell line expressing a high level of BAALC, such as Kasumi-1 cells. Decelerated cell cycling was also observed in the BAALC-knocked down cells. Notably, the proportion of the phosphorylated ERK bound to MEK was decreased when BAALC was knocked down, which is consistent with BAALC-overexpressed experiments. Interestingly, in AML cell lines without active Ras mutations, the expression of BAALC was positively correlated to its sensitivity to MEK inhibitor (U0126). Moreover, growth advantage of BAALC overexpression in leukemic cell lines was canceled by the treatment with U0126, implying that BAALC-induced growth advantage depends on MEK-ERK signaling activity. Analysis of deletion mutants of BAALC revealed that binding ability of BAALC to MEK resides in its N-terminal region, which is highly conserved among mammals. This N-terminal region of BAALC was necessary for phosphorylation of ERK and the mutant lacking this region failed to promote proliferation of the leukemic cell lines. Taken together, BAALC acts as a scaffold-like protein that binds to MEK in the cytoplasm and directly up-regulates MEK-ERK signal transduction pathway. The fact that overexpression of BAALC significantly promotes proliferation of leukemic cells is consistent with the clinical observation that BAALC-high leukemia has a poor prognosis. The novel function of BAALC as a scaffold-like protein that activates MEK-ERK signal transduction pathway may account for the underlying mechanism for the generation of aggressive form of leukemia and would be a promising therapeutic target of BAALC-high leukemia. Disclosures:Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.